Method for the treatment or prophylaxis of lymphangioleiomyomatosis (lam) and animal model for use in lam research

ABSTRACT

Treatment of lymphangioleiomyomatosis with the MEK1/2 inhibitor CI-1040 delayed the development of primary tumors and blocked the estrogen-induced lung metastases in treated animals. Such treatment also reduced the number of circulating ELT3 cells and decreased their lung colonization after intravenous injection.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 12/989,529, filed Oct. 25, 2010, which is the U.S.national stage of International Application No. PCT/US2009/044643, filedMay 20, 2009 and claims the benefit of U.S. Provisional PatentApplication No. 61/054,714, filed May 20, 2008, each of the entiredisclosures of which is incorporated by reference herein.

STATEMENT REGARDING FEDERAL SPONSORED RESEARCH OR DEVELOPMENT

The present invention was made with funds provided by the NationalInstitute of Health under Grant No. HL 60746. The U.S. Government hascertain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to the treatment or prophylaxis oflymphangioleiomyomatosis (LAM) in mammals, by the administration ofcertain inhibitors of the dual-specificity kinases MEK-1 and MEK-2, aswell as a novel animal model useful in the development of potentialtargeted therapies for LAM and other estrogen-mediated malignancies.

BACKGROUND OF THE INVENTION

LAM, the pulmonary manifestation of tuberous sclerosis complex (TSC), isan often-fatal disease which is characterized by the widespreadproliferation of abnormal smooth muscle cells that grow aberrantly inthe lung, producing cystic changes within the lung parenchyma (Sullivan,E. J., Chest, 114: 1689-1703 (1998)). For reasons that are not clearlyunderstood, LAM affects women almost exclusively. LAM affects 30-40% ofwomen with TSC (Costello, L. C. et al., Mayo Clin. Proc. 75: 591-594(2000); Franz, D. N. et al., Am. J. Respir. Crit. Care Med., 164:661-668 (2001)). LAM can also occur, in women who do not have clinicalmanifestations of TSC, as well as those who do not have germlinemutations in TSC1 or TSC2 (sporadic LAM).

The lungs in LAM are diffusely infiltrated by histologically benign,immature-appearing smooth muscle cells that express estrogen receptor(ER) alpha and progesterone receptor. This cellular infiltration isaccompanied by cystic lung degeneration. Most women with TSC-associatedLAM and 60% of women with sporadic LAM have renal angiomyolipomas(AMLs), which contain abnormal smooth muscle cells that are virtuallyidentical to LAM cells. The relentless growth of LAM cells in thepulmonary airway, parenchyma, lymphatics and blood vessels leads torespiratory failure and death. In a Mayo Clinic series, LAM was thethird most frequent cause of TSC-related death, after renal disease andbrain tumors (Shepherd, C. W. et al., Mayo Clin. Proc., 66: 792-796(1991)).

Genetic studies by the present inventors and others have revealed thatLAM cells from both TSC-LAM and sporadic LAM carry inactivatingmutations in both alleles of the TSC1 or TSC2 genes, and spread to thelungs via a metastatic mechanism despite the fact that LAM cells have ahistologically benign appearance. Genetic evidence for this “benignmetastasis” model of LAM has arisen from women with the sporadic form ofLAM, who have somatic TSC2 mutations in LAM cells and renal AML cellsbut not in normal kidney, lung, or peripheral blood cells (Yu, J. etal., Am. J. Respir. Crit. Care Med., 164: 1537-1540 (2001); Carsillo,T., Proc. Natl. Acad. Sci. USA, 97: 6085-6090 (2000)); and fluorescentin situ hybridization analysis of LAM that recurs after lungtransplantation (Karbowniczek, M. et al., Am. J. Respir. Crit. CareMed., 167: 976-982 (2003)). The presence of disseminated neoplasticcells has been detected in blood and body fluids from LAM patients(Crooks, D. M. et al., Proc. Natl. Acad. Sci. USA, 101: 17462-17467(2004)).

The protein products of TSC1 and TSC2, hamartin and tuberin,respectively, form heterodimers (Plank, T. L. et al., Cancer Res., 58:4766-4770 (1998); van Slegtenhorst, M. et al., Hum. Mol. Genetc., 7:1053-1057 (1998)) that inhibit the small GTPase Ras homologue enrichedin brain (Rheb), via tuberin's highly conserved GTPase activating domainIn its active form, Rheb activates the mammalian target of rapamycin(mTOR) complex 1 (TORC1), which is a key regulator of proteintranslation, cell size, and cell proliferation (Crino, P. B., N. Engl.J. Med., 355: 1345-1356 (2006). Evidence of OTRC1 activation, includinghyperphosphorylation of ribosomal protein S6, has been observed in tumorspecimens from TSC patients and LAM patients (El-Hashemite, N. et al.,Lancet, 361: 1348-1349 (2003); Karbowniczek, M. et al., Am. J. Pathol.,162: 491-500 (2003); Yu. J., Am. J. Physiol. Lung Cell Mol. Physiol.,286: L694-700 (2004)). Independent of its activation of mTOR, Rhebinhibits the activity of B-Raf and C-Raf/Raf-1 kinase, resulting inreduced phosphorylation of p42/44 MAPK (Im. E. et al., Oncogene, 21:6356-6365 (2002); Karbowniczek, M. et al., J. Biol. Chem., 279:29930-29937 (2004); Karbowniczek, M. et al., J. Biol. Chem., 281:25447-25456 (2006)), but the impact of the Raf/MEK/MAPK pathway ondisease pathogenesis is undefined.

The female predominance of LAM, coupled with the genetic data indicatingthat LAM cells are metastatic, suggests that estrogen may promote themetastasis of tuberin-null cells. Both LAM cells and angiomyolipomacells express estrogen receptor alpha (Logginidou, H. et al., Chest.,117: 25-30 (2000)), and there are reports of symptom mitigation in LAMpatients after oophorectomy and worsening of symptoms during pregnancy(Sullivan, E. J. et al., supra). However, the molecular and cellularmechanisms that may underlie an impact of estrogen on the metastasis ofLAM cells are not well defined, in part because of the lack of in vivomodels that recapitulate the metastatic behavior of LAM cells.

Oxygen therapy may become necessary if the disease continues to worsenand lung function is impaired. Lung transplantation is considered as alast resort.

Although the immunosuppressant drug sirolimus (rapamycin) has shownpreliminary promise as a potential LAM therapy, there is no currentlyapproved drug for the treatment or prophylaxis of LAM.

New therapies and preventatives are clearly needed for LAM and relatedpathologies of similar etiology.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there isprovided a method for the treatment or prophylaxis oflymphangioleiomyomatosis (LAM) comprising administering to a patient inneed of such treatment or prophylaxis a therapeutically effective amountof a compound having the formula:

wherein:

R₁ is hydrogen, hydroxy, C₁-C₈ alkyl, C₁-C₈ alkoxy, halo,trifluoromethyl, or CN;

R₂ is hydrogen;

R₃, R₄, and R₅ independently are hydrogen, hydroxy, halo,trifluoromethyl, C₁-C₈ alkyl, C₁-C₈ alkoxy, nitro, CN, or (O orNH)_(m)—(CH₂)_(n)—R₉, where R₉ is hydrogen, hydroxy, CO₂H or NR₁₀R₁₁;

n is 0 to 4;

m is 0 or 1;

R₁₀ or R₁₁ independently are hydrogen or C₁-C₈ alkyl, or taken togetherwith the nitrogen to which they are attached can complete a 3- to10-member cyclic ring optionally containing one, two, or threeadditional heteroatoms selected from O, S, NH, or N—C₁-C₈ alkyl;

R₆ is hydrogen, C₁-C₈ alkyl,

alkyl, aryl, aralkyl, or C₃-C₁₀ cycloalkyl;

R₇ is hydrogen, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,

C₃-C₁₀ (cycloalkyl or cycloalkyl optionally containing a heteroatomselected from O, S, or NR₉);

X is bromine or iodine;

and wherein any of the foregoing alkyl, alkenyl, and alkynyl groups canbe unsubstituted or substituted by cycloalkyl (or cycloalkyl optionallycontaining a heteroatom selected from O, S, or NR₉), aryl, aryloxy,heteroaryl, or heteroaryloxy; or R₆ and R₇ taken together with the N—Oto which they are attached can complete a 5- to 10-membered cyclic ring,optionally containing one, two, or three additional heteroatoms selectedfrom O, S, or NR₁₀R₁₁.

In another aspect, the present invention provides a xenograft rodentmodel using TSC2-deficient rat uterine lecomyoma (ELT3) cells, whichupon subcutaneous inoculation into CB17-scid mice and subsequentadministration of estrogen, develop tumors and exhibit pulmonarymetastases. This animal model serves as a useful tool for theidentification of agents having therapeutic efficacy for the treatmentof LAM, and other hormonally-driven conditions.

As will appear in the following description, the research underlyingthis invention revealed that the MEK pathway is a critical component ofthe estrogen-dependent metastatic potential of Tsc2-null cells, and leadto a unique model of LAM pathogenesis with therapeutic implications inwhich E₂ promotes the survival of disseminated LAM cells, therebyfacilitating lung colonization and metastasis. See Yu, J. J. et al.,PNAS, 106(8): 2635-2640 (2009) 15

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of various experiments indicating that estrogenpromotes the lung metastasis of tuberin-deficient ELT3 cells in femaleand male mice, including:

FIG. 1A—The proliferation of ELT3 cells in response to E2 was measuredby 3H-thymidine incorporation after 5 days of growth;

FIG. 1B—Lung metastases were scored from E2 (n=9) or placebo-treated(n=10) mice;

FIG. 1C—The number of lung metastases in male mice was scored fromplacebo (n=10) and E2-treated (n=9) mice;

FIG. 1D-FIG. F—Consecutive lung sections containing metastases (arrows)from an E2-treated female mouse were stained with hematoxylin and eosin(H&D) (FIG. 1D); anti-smooth muscle actin (FIG. 1E); and anti-phospho-S6(FIG. 1F) (scale bar, 50 μM)

FIG. 1G—Anti-phospho-S6 immunostain of the primary xenograft tumor of anestrogen-treated female mouse.

FIG. 1H—Phospho-S6 immunoreactivity of a metastasis of anestrogen-treated male mouse;

FIG. 1I—Phospho-S6 immunoreactivity of a xenograft tumor of anestrogen-treated male mouse (scale bar, 20 μM).

FIG. 2 shows the results of experiments indicating that estrogenincreases circulating tumor cells in mice bearing xenograft tumors andenhances the survival and lung seeding of intravenously injectedTsc2-null cells, including:

FIG. 2A—DNA prepared from the blood of placebo (n=3) and E₂-treated(n=3) mice bearing xenograft tumors of similar size (≈1,000 mm³) wasanalyzed by real-time PCR using rat-specific primers to quantitatecirculating tumors;

FIG. 2B—Levels of circulating tumor cell DNA 6 h after i.v. injection ofELT3 cells into placebo (n=3) and E₂-treated (n=3) mice; and

FIG. 2C—Levels of tumor cell DNA in the lungs 24 h after i.v. injectionof ELT3 cells into placebo (n=3) and E₂-treated (n=3) mice.

FIG. 3 shows the results of experiments indicating that estrogenpromotes the lung colonization of Tsc2-null ELT3 cells, including:

FIG. 3A—ELT3-luciferase cells were injected intravenously intooveriectomized female placebo (n=3) and E₂-treated (n=3) mice. Lungcolonization was measured using bioluminescence at 1, 3, and 24 h afterinjection. Representative images are shown;

FIG. 3B—Total photon flux/second present in the chest regions in placebo(n=3) and E₂-treated (n=3) animals.

FIG. 3C—Lungs were dissected 24 h postcell injection and bioluminescencewas imaged in Petri dishes.

FIG. 4 shows the results of experiments indicating that estrogenactivated p42/44 MAPK in ELT3 cells in vitro and in vivo, including:

FIG. 4A—Levels of phosphorylated p42/44 MAPK and total MAPK weredetermined by immunoblot analysis. Pretreatment with PD98059 blockedE₂-induced MAPK activation. [3-Actin immunoblotting was included as aloading control;

FIG. 4B—Levels of phosphorylated C-Raf/Raf-1 and total Raf-1 after E₂stimulation;

FIG. 4C—Levels of phosphorylated S6 after E2 stimulation;

FIG. 4D—The nuclear and cytoplasmic fractions were separated, and levelsof phosphor-p42/44 MAPK were examined by immunoblot analysis. Anti-ELK1and anti-α-tubulin were included as loading controls for the nuclear andcytosolic fractions, respectively;

FIG. 4E-FIG. 4F—Pulmonary metastases from an E₂-treated mouse showedhyperphosphorylation of p42/44 MAPK (scale bar, 50 μM and 125 μM);

FIG. 4G-FIG. 4H—Phospho-p42/44 MAPK (T202/Y204) immunostaining ofprimary tumor sections from placebo-treated mice (FIG. 4G) andE2-treated mice (FIG. 4H); and

FIG. 4I—Percentage of cells with nuclear immunoreactivity ofphosphor-p42/44 MAPK was scored from 4 random fields per section.

FIG. 5 shows the results of experiments indicating that estrogenincreases the resistance of ELT3 cells to anoikis, including;

FIG. 5A—The level of cleaved caspase-3 was determined by immunoblotanalysis. α-Tubulin is included as a loading control;

FIG. 5B—DNA fragmentation was assessed by ELISA;

FIG. 5C—Cell growth was measured by 3H-thymidine incorporation after 24h of growth on PolyHEMA plates in the presence or absence of E₂,followed by 24 h of growth on adherent plates in the absence of E₂; and

FIG. 5D—Levels of phosphor-p42/44 MAPK, MAPK, Bim, cleaved caspase-3,phosphor-S6K, and phosphor-S6 were determined by immunoblot analysis.α-Tubulin is included as a loading control.

FIG. 6 shows the results of experiments indicating that the MEK 1/2inhibitor CI-1040 blocks the estrogen driven metastasis of ELT3 cells invivo. In these experiments, ELT3 cells were injected into femaleovariectomized nude mice implanted with estrogen or placebo pellets. InFIG. 6A-FIG. 6E, animals were treated with CI-1040 (150 mg/kg/day bygavage, twice a day) starting 1 day post-ELT3 cell inoculation for thexenograft experiments.

FIG. 6A—Tumor development was recorded as the percentage of tumor-freeanimals post-cell inoculation;

FIG. 6B—The primary tumor area was calculated at 7 weeks post-cellinoculation;

FIG. 6C—The level of circulating ELT3 cells was measured from bloodsamples of xenograft animals using rat-specific qPCR amplification;

FIG. 6D—The percentage of mice with lung metastases in the placebo andestrogen-treated groups was compared;

FIG. 6E—The number of lung metastases was scored;

FIG. 6F—ELT3-luciferase cells were injected intravenously intooveriectomized female E₂-treated (n=5) and CI-1040 plus E₂-treated (n=5)mice. CI-1040 was administered according to the same regimen starting 2days before cell inoculation. Lung colonization was measured usingbioluminescence 2 and 5 h after injection. Total photon flux/secondpresent in the chest regions were quantified and compared between E₂(n=5) and CI-1040 plus E₂-treated (n=5) animals. Lungs were dissectedand imaged 60 h post-cell injection. Total photon flux/second present inex vivo lungs were quantified and compared between E₂ (n=5) and CI-1040plus E₂-treated (n=5) animals.

FIG. 7 shows the mTOR inhibitor RAD001 blocks primary tumor developmentand estrogen-driven metastasis of ELT3 cells in vivo. ELT3 cells wereinjected into female overiectomized nude mice implanted with estrogen orplacebo pellets Animals were treated with RAD001 (4 mg/kg/day by gavage)starting 1 day post-ELT3 cell inoculation. In FIG. 7A, the primary tumorarea was calculated at 8 weeks post-cell inoculation. In FIG. 7B, thenumber of lung metastases was scored at 8 weeks post-cell inoculation.*, P<0.05, Student's t test.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on important insights gainedfrom the discovery that 17β-estriadiol (E2) promotes the pulmonarymetastases of tuberin-deficient ELT3 cells, associated with activationof p42/44 mitogen-activated protein kinase (MAPK), elevated number ofcirculating tumor cells and prolonged survival of intravenously injectedELT3 cells. In other words, estrogen was found to induce thedissemination of tumor cells, increase the number of circulating tumorcells and enhance lung colonization. This discovery suggested severalregimens for possible therapeutic intervention against LAM, includingtreatment with an inhibitor of the dual specificity kinases, MEK-1 andMEK-2. The latter approach was found to delay the development of primarytumors and block estrogen-induced lung metastases in animals Inhibitionof MEK1/2 also reduced the number of circulating ELT3 cells anddecreased their lung colonization after intravenous injection.

The MEK1/2 inhibitor that produced these results, namely,2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide(also known as CI-1040), is from a class of compounds referred to asphenylamino-benzhydroxamic acid derivatives, which are represented byFormula I, above.

In view of the mechanism of action of CI-1040, i.e., inhibition of themitogen activated extracellular signal regulated kinase (ERK) pathwaywhich is known to be involved in key cellular activities includingproliferation, differentiation, apoptosis and angiogenesis, it isbelieved that analogous compounds within the scope of Formula I, above,will exhibit similar therapeutic activity against LAM.

The synthesis of phenylamino-benzhydroxamic acid derivatives of FormulaI, above, and the preparation of pharmaceutical compositions comprisingsuch derivatives are described in detail in International PatentApplication No. PCT/US98/13106 (WO 99/01426) and U.S. Pat. No.6,821,963.

A suitable route of administration and maximum tolerable dose foradministration of CI-1040 to humans have been determined in a phase Iand pharmacodynamic study (LoRusso (2005)).

The therapy described herein will typically be administered for a periodof time sufficient to provide an appreciable improvement orstabilization of lung function in the patient undergoing treatment, asdetermined by a reduction in lung metastasis and/or a reduction in tumorburden. As used herein, the term “patient” refers to animals, includingmammals, preferably humans.

The term “anoikis” as used herein refers to matrix deprivation-inducedapoptosis which is a form of programmed cell death induced whenanchorage-dependent cells detach from the surrounding extracellularmatrix (ECM). The ECM provides essential signals for cell growth orsurvival. When cells are detached from the ECM, i.e. there is a loss ofnormal cell-matrix interactions, they may undergo anoikis. Metastatictumor cells are often resistant to anoikis and invade other organs.

While not wishing to be confined to any particular theory regarding themechanism responsible for the observed therapeutic effect that thecompounds of Formula I have on LAM, it is believed that the compoundsdisrupt key signaling events associated with angiogenesis and cellularproliferation.

Based on the suspected mechanism of action of the compounds of FormulaI, as mentioned above, it is anticipated that these compounds will beuseful not only for therapeutic treatment of LAM, but for prophylacticuse as well. The dosages may be essentially the same, whether fortreatment or prophylaxis of LAM.

Applications of the compounds described herein may extend to otherestrogen-mediated malignancies by modulating MEK/MAPK signalling. It hasbeen shown, for example, that estrogen enhances liver hemangiomadevelopment in Tsc±mice (El-Hashemite et al., Cancer Res., 65: 2474-2481(2005)).

The following examples describe the invention in further detail. Theseexamples are provided for illustrative purposes only and should in noway be construed as limiting the invention.

Materials and Methods Cell Culture and Reagents

ELT-3 cells (Eker rat uterine leiomyoma-derived smooth muscle cells)were cultured in IIA complete medium (DMEM/F12 basal media including 15%FBS, 0.2 μM hydrocortisone, 10 μU/mL vasopressin, 1× FeSO4, 10 ng/mLEGF, 1× ITS, 0.01 nM triiolythryonine, 0.12% sodium bicarbonate, 1×cholesterol and 1× penicillin/streptomycin) supplemented with 15% FBS.Prior to the in vitro experiments, cells were maintained in mediasupplemented with 10%-charcoal-stripped FBS for three days and thenserum starved for 24 hours in serum-free and phenol red-free medium. E2(10 nM, Sigma, St. Louis, Mo.) or PD98059 (50 μM, Cell SignalingTechnology, Danvers, Mass.) was added to the cells as indicated.

Immunoblotting and Antibodies

Cells were rinsed once in ice-cold PBS and lysed in PTY buffer (50 mMHepes, pH 7.5, 50 mM NaCl, 5 mM EDTA, 50 mM NaF, 10 mM Na₄P₂O₇, and 1%Triton 100) supplemented with phosphatase inhibitors. Lysates wereresolved by SDS-PAGE electrophoresis and transferred onto Immobilon Pmembranes (Millipore). Cytoplasmic and nuclear fractions were separatedusing CellLytic Nuclear Extraction Kit (Sigma) before electrophoresis.The following antibodies were used for Western blot analysis: anti-Bim(Affinity BioReagents), anti-S6, anti-phospho-S6 (S235/236),anti-phospho-S6K (T389), anti-phospho-p42/44 MAPK (T202/Y204),anti-p42/44MAPK, anti-ELK1, anti-cleaved caspase 3 (all from CellSignaling technology), anti-Ki67 and antismooth muscle actin(Bio-Genex), anti-alpha-tubulin and anti-beta-actin (Sigma),anti-phospho-Raf-1 (S338) (Upstate Biotechnology), and anti-Raf-1 (SantaCruz Biotechnology). Western blots were developed using horseradishperoxidase-conjugated secondary antibodies and ECL chemiluminescence(Amersham Biosciences).

Immunohistochemistry

Sections were deparaffinized, incubated overnight with primaryantibodies at 4° C. in a humidified chamber and then rinsed andincubated with biotinylated secondary antibodies for 30 minutes at roomtemperature. Slides were developed using the Broad Spectrum AECHistostain-Plus (Invitrogen) or Histostain-Plus kit (Invitrogen), andthey were counterstained with Gill's hematoxylin.

Animal Studies

All animal work was performed in accordance with a protocol approved bythe FCCC Institutional Animal Care and Use Committee. Male CB-17 scidmice, six weeks of age, were purchased from Fox Chase Cancer Center.Female ovariectomized CB 17-scid mice, six to eight weeks of age, werepurchased from Taconic (Hudson, N.Y.). One week prior to cell injection,17-beta estradiol or control-placebo pellets (2.5 mg, 90-day release)(Innovative Research America, Sarasota, Fla.) were implanted. Forxenograft tumor establishment, subconfluent ELT3 cells were harvested,washed in PBS, and resuspended in 0.2 ml of serum-free medium. Twomillions ELT3 cells were injected into both flanks of the mousesubcutaneously. For metastatic and circulating tumor cells assays, 2×105 ELT3 or ELT3-Luc cells were resuspended in 0.1 ml PBS and injectedinto the lateral tail vein of mice. Lung metastases were scored from 3-5five-micron H&E stained sections of each lobe at 40× magnification byobservers blinded to the experimental conditions.

Pharmacological Inhibitor

CI-1040 (PD184352) was obtained from Pfizer (Ann Arbor, Mich.) and wasprepared in a vehicle of 10% Cremophore EL (Sigma), 10% ethanol and 80%water. RAD001 was obtained from Novartis Pharma AG (Basel, Switzerland)and was diluted in double-distilled water. Drug were administered oneday post cell inoculation at the following doses: RAD001 (4mg/kg/day,gavage); CI-1040, (150 mg/kg/day gavage, twice a day)

Xenograft Tumor Samples

Subcutaneous tumors were removed from animals upon sacrifice. Tumorweights were recorded, and tumor size was measured in two dimensionswith calipers.

Detection of ELT Cells by Real-Time PCR

Mouse blood (0.5 mL) was collected at indicated times by intraocularbleed, and red blood cells were lysed before DNA extraction. At death,the lungs were dissected and stored at −80° C. for DNA extraction. Ratand mouse DNAs were quantified by using TaqMan-chemistry based real-timePCR assays. The assay for rat DNA was adapted from the method describedby Walker et al., Genomics, 83: 518-527 (2004). The primers amplify aLINE repeat element (AC087102). The assay for mouse is for the gene Anf.The sequences (all 5′ to 3′) for the primers and probes are:

Mouse Forward: GGCATCTTCTGCTGGCTCC; Reverse: GGCTA GAACCCTCCCCATTCT;Probe: 6FAM-CACTCCATCGCTTATCGCTGCAAGTG-BHQ1. Rat Forward:CAAGACGGATGATCAAAATGTG; Reverse: TCTCTGTTTTAATCTTTGCCT CTCC; Probe:6FAM-CCTGCCAAGGGTATTCTTTTTCCTCATTTA AA-BHQ1.PCR master mix from Eurogentec was used for PCR. Primers and probeconcentrations were 500 and 100 nM, respectively. Cycling conditionswere 95° C., 15 minutes followed by 40 (2-steps) cycles (95° C., 15 sec;60° C., 60 sec). Reactions were run using an ABI 7900 HT instrument.Each sample was analyzed using two different amounts of input DNA.Relative quantification was done using the 2^(−ΔΔCt) method (Livak. K.J., Methods, 25: 402-408 (2001)).

Bioluminescent Reporter Imaging

One million ELT3 cells were transfected with 3 μg of pCMV-Luc(Invitrogen, Carlsbad, Calif.) using Nucleofection reagent (Amaxa,Gaithersburg, Md.). Cells were selected in G418 for 2 weeks, and G418resistant clones were isolated and examined for luciferase activity.

Ten minutes prior to imaging, animals were injected with luciferin(Xenogen, Alameda, Calif.) (120 mg/kg, i.p.). Bioluminescent signalswere recorded at indicated times post cell injection using Xenogen IVISSystem (Xenogen). Total photon flux at the chest regions and from thedissected lungs was analyzed.

Anoikis Assay

ELT3 cells were cultured with or without 10 nM E₂ in serum-free andphenol red-free medium supplemented with 10% charcoal-stripped FBS for24 hours. Cells were harvested, plated onto 60×15 mm stylePoly-hydroxyethyl methacrylate (PolyHEMA) culture dishes (ComingIncorporated) at a density of 1×606 cells/mL with or without E₂. Celldeath as a function of DNA fragmentation was detected using Cell DeathDetection ELISA kit (Roche Diagnostics).

Thymidine Incorporation

The surviving cells in suspension were plated in triplicate in 24-wellplates and allowed to grow adherently for 24 hours. ³H-thymidine (1 μCi)was added to the media and the cells were incubated at 37° C. for 6hours, washed with PBS, and lysed in 0.5 mL of 0.5 N NaOH plus 0.5% SDS.³H-thymidine incorporation was measured by scintillation counting.

Statistical Analysis

Statistical analyses were performed using Student's t test whencomparing 2 groups. Results are presented as means±SD of experimentsperformed in triplicate. Differences were considered significant atP<0.05 (*).

Results Estrogen Promotes Pulmonary Metastasis of Tuberin-Deficient ELT3Cells in Ovariectomized Female and Male Mice

To study the role of E₂ in the metastasis of Tsc2-null cells, ELT3 cellswere used, which were originally derived from a uterine leiomyoma in theEker rat model of Tsc2 and, similar to LAM cells, express smooth musclecell markers and estrogen receptor alpha (Howe et al., Am. J. Pathol.,146: 1568-1579 (1995); Howe, S. R. et al., Endocrinology, 136: 4996-5003(1995)). To confirm that ELT3 cells proliferate in response to estrogenstimulation in vitro, cell growth was measured using ³H-thymidineincorporation. E₂ treatment resulted in a significant increase in³H-thymidine incorporation by 2.8-fold on day 5 (P=0.03, FIG. 1A),similar to the findings of Howe et al., Endocrinology, supra (1995)).

ELT3 cells were inoculated subcutaneously into the flanks ofovariectomized CB17-SCID mice, which were supplemented 1 week beforewith either placebo or E₂ pellets (2.5 mg, 90-day release). Tumors arosein 100% of both estrogen and placebo-treated mice. At post-inoculationweek 8, estrogen-treated mice had a mean tumor area of 287±43 mm²,whereas placebo-treated mice had a mean tumor area of 130±20 mm²(P=0.0035), consistent with previous findings (Howe et al.,Endocrinology, supra (1995)). The proliferative potential of ELT3 cellsin vivo was examined using Ki-67 immunoreactivity. The number of Ki-67positive cells in estrogen-treated tumors was 17% higher than the numberin placebo-treated tumors (P=0.03).

Pulmonary metastases were identified in 5 of 9 E₂-treated mice (56%),with an average of 15 metastases/mouse (range 4-37) (FIG. 1B). Incontrast, only 1 of 9 placebo-treated mice (10%) developed a singlemetastasis (P=0.039). To determine whether the enhanced metastasis wasdirectly related to tumor size, a subset of placebo-treated mice (n=4)and estrogen-treated mice (n=4) that developed primary tumors at similarsize (209±16 and 198±20 mm², respectively) was analyzed separately.Three of the estrogen-treated mice developed pulmonary metastases withan average of 6 metastases/mouse, while none of the placebo-treated micedeveloped metastases.

Next, ELT3 cells were inoculated into male mice. At 8 weeks post-cellinoculation, E₂-treated animals developed tumors that were 2.9-foldlarger than those in the placebo-treated animals. As in the female mice,E₂ significantly enhanced the frequency and the number of pulmonarymetastases. At 8 weeks post-inoculation, 10 of 10 (100%) of theE₂-treated mice developed metastases, with an average of 14metastases/mouse (range 5-32). In contrast, 7 of 10 (70%) of theplacebo-treated mice developed metastases, with an average of 4metastases/mouse (range 1-7, P=0.013) (FIG. 1C). As expected, themetastatic and primary tumor cells were immunoreactive for smooth muscleactin and phosphor-ribosomal protein S6 (FIG. 1D-I).

Inhibition of mTOR Blocks Estrogen-Induced Pulmonary Metastasis ofTsc2-Null Cells

To determine the role of mTOR signaling pathway in the estrogen-inducedmetastasis of tuberin-deficient ELT3 cells, the mTORC1 inhibitor RAD001(4 mg/kg/day by gavage) was administered 5 days per week beginning 1 daypost-cell inoculation. RAD001 completely blocked both primary tumordevelopment (FIG. 7A) and lung metastasis (FIG. 7B) in the presence ofestrogen or placebo.

Estrogen Increases the Number of Circulating Tumor Cell DNA

To determine whether the mechanism of E₂-driven metastasis of ELT3 cellsis associated with an increase in survival of ELT3 cells in thecirculation, we analyzed blood collected from xenograft mice at 7 weekspost-cell inoculation. Real-time PCR with rat-specific primers was usedto measure the relative quantity of tumor cells circulating in theblood. We selected 6 animals (3 placebo, 3 E₂-treated) bearing tumors ofsimilar size (≈1,000 mm³) for this analysis. The E₂-treated animals hada striking increase in the amount of circulating tumor cell DNA ascompared to that in the placebo-treated animals (P=0.034, FIG. 2A).

This increased level of circulating tumor cell DNA suggested that E₂ maypromote the survival of Tsc2-null cells upon dissemination from theprimary tumor site. To test this, we injected 2×10⁵ ELT3 cellsintravenously and again measured the amount of tumor cell DNA usingreal-time PCR. E₂ treatment results in a 2.5-fold increase incirculating cells 6 h post-injection (P=0.047, FIG. 2B). To determinewhether this enhanced survival of circulating cells was associated withincreased colonization of the lungs, the mice were killed 24 h afterinjection, and the lungs were analyzed by real-time PCR. E₂ treated micehad a 2-fold increase in the lung seeding of ELT3 cells (P=0.039, FIG.2C).

Estrogen Promotes the Lung Colonization of ELT3 Cells In Vivo

To identify the earliest time points at which estrogen exerts an effecton the survival of intravenously injected Tsc2-null cells, ELT3 cellsthat stably express luciferase (ELT3-Luc) were intravenously injected.The level of bioluminescence was evaluated using the Xenogen IVISSystem. at 1 h post-cell injection, similar levels of bioluminescencewere observed in the chest regions of E2 and placebo-treated mice. by 3h, the bioluminescence in the chest regions was 2-fold higher in theE2-treated animals than in the placebo-treated animals, and at 24 hpost-cell injection it was 5-fold higher in the E2-treated animals(P=0.043, FIGS. 3A and B). After sacrifice, the lungs were dissected andimaged in Petri dishes to confirm that the bioluminescent signals in thechest regions of the living mice were a result of lung colonization(FIG. 3C).

Estrogen Activates p42/44 MAPK in ELT3 Cells In Vivo and In Vitro

These results suggested that E₂ promotes the survival of disseminatedELT3 cells. To determine the mechanism of this, we focused on theRaf/MEK/MAPK signaling cascade. This pathway is inhibited in cellslacking TSC2 via Rheb's inhibition of B-Raf and C-Raf/Raf-1 kinase (13,14). E₂ has been shown to activate p42/44 MAPK in ELT3 cells and in LAMpatient-derived cells (Yu, J. et al., supra (2004); Finlay, G. A. etal., Am. J. Physiol. Cell Physiol., 285: C409-418 (2003); Finlay, G. A.et al., J. Biol. Chem., 279: 23114-23122 (2004)). To confirm that E₂activated MAPK in ELT3 cells, cells were treated with 10 nM E₂ andexamined the phosphorylation status of p42/44 MAPK by immunoblotting.Within 15 min, E₂ induced the phosphorylation of p42/44 MAPK (FIG. 4A).It was also found that E₂-induced phosphorylation of p42/44 MAPK wasblocked by the MEK1/2 inhibitor PD98059 (FIG. 4A), which is in contrastto the prior work of Finlay et al., supra. E₂ is known to rapidlyactivate C-Raf (Pratt, M. A. et al., Mol. Cell Biochem, 189: 119-125(1998)). It was hypothesized that E₂ reactivates MAPK via aRheb-independent pathway in cells lacking tuberin. In a separateexperiment, it was found that E₂ rapidly (within 2 min) increased thephosphorylation of C-Raf at Ser-338, a site which is closely linked withC-Raf activity (FIG. 4B). However, E₂ does not affect mTOR activation asmeasured by ribosomal protein S6 phosphorylation (FIG. 4C). Theseresults suggest that E₂ does not regulate Rheb activity and that thepotential of E₂ to impact the Raf/MEK/ERK kinase cascade is Rhebindependent. Nuclear translocation of phospho-MAPK was observed within 5min of E₂ exposure (FIG. 4D).

These in vitro findings led us to examine whether E₂ activates P42/44MAPK in ELT3 cells in vivo. In lungs from E₂-treated animals, nuclearphosphor-p42/44 MAPK staining was observed in metastases but not inadjacent normal tissues (FIGS. 4E and F). In the primary xenografttumors, the percentage of cells with primarily nuclear phospho-MAPK wassignificantly higher in the tumors from the E₂-treated animals, comparedto the tumors from placebo-treated animals (65% vs. 28%, P=0.001, FIG.4G-I).

Estrogen Increases the Resistance of ELT3 Cells to Anoikis In Vitro

These in vivo findings suggest that estrogen enhances the survival ofcirculating tumor cells in a MAPK-dependent manner. Because detachedcells normally undergo apoptosis (Reginato, M. J. et al., Nat. CellBiol., 5: 733-740 (2003); Rytomaa, M. et al., Curr. Biol. 9: 1043-1046(1999); Schulze, A. et al., Genes Dev., 15: 981-994 (2001)), a criticalfirst step in cancer progression is the development of resistance tomatrix deprivation-induced apoptosis (anoikis) (Hanahan, D. et al.,Cell, 100: 57-70 (2000); Eckert, L. B. et al., Cancer Res., 64:4585-4592 (2004)). Therefore, to investigate the mechanism ofE₂-prolonged survival of ELT3 cells in the circulation, we examined theeffect of estrogen on anoikis. ELT3 cells were treated for 24 h witheither 10 nM E₂ or control and then plated onto PolyHEMA, which preventsattachment and therefore induces anoikis. Cell lysates wereimmunoblotted for cleaved caspase-3, which is a measure of apoptosis. E₂treatment reduced caspase-3 cleavage at 6, 16 and 24 h (FIG. 5A). E₂treatment also significantly reduced DNA fragmentation at 1 and 24 h(P=0.001 and P=0.015, FIG. 5B), which indicates that E2 inhibits anoikisof Tsc2-null cells.

To confirm further that E₂ promotes the survival of detached cells, ELT3cells were plates onto PolyHEMA plates for 24 h and replated onto normaltissue culture dishes. Cell growth was measured using ³H-thymidineincorporation. E₂ treatment results in a significant increase in³H-thymidine incorporation 24 h after replating (P=0.008, FIG. 5C). ThisE₂-enhanced survival was blocked by treatment with MEK1/2 inhibitorPD98059 (P=0.035, FIG. 5C).

To determine the components that mediate estrogen-enhanced resistance ofELT3 cells to anoikis, we analyzed the proapoptotic protein, Bcl-2interacting mediator of cell death (Bim), which is known to be acritical activator of anoikis (Reginato, M. J. et al., supra). Bim isphosphorylated by protein kinases, including p42/44 MAPK, which leads torapid proteasomal-mediated degradation and increased cell survival (Tan,T. T. et al., Cancer Cell, 7: 227-238 (2005)). Bim protein level wasexamined by immunoblotting. We found that estrogen decreased theaccumulation Bim after 1 h in detachment conditions (FIG. 5D).Preincubation with the MEK inhibitor PD98059 partially blockedestrogen's inhibition of Bim accumulation and capase-3 cleavage after 4h in detachment conditions (FIG. 5D). We also examined thephosphorylation of S6K and S6 in detachment conditions and found thatthe phosphorylation of S6K and S6 did not change with E₂ stimulation.Interestingly, treatment with PD98059 decreased the phosphorylation ofS6K 1 h after detachment (FIG. 5D).

The MEK1/2 Inhibitor CI-1040 Blocks the Estrogen-Driven Metastasis ofELT3 Cells In Vivo

These in vitro and in vivo results suggest that E₂-induced activation ofthe MEK/MAPK pathway contributes to the metastatic potential ofcirculating Tsc2-null ELT3 cells. To determine the effect of inhibitingthe MEK/MAPK pathway on the pulmonary metastasis of Tsc2-null cells invivo, we used the inoculation of ELT3 cells, animals, implanted witheither placebo or estrogen pellets, were treated with CI-1040 (150 mg/kgday by gavage, twice a day) (Sebolt-Leopold, J. S. et al., 5: 810-816(1999)). CI-1040 delayed tumor formation (FIG. 6A) and reduced the sizeof primary tumors by 25% in E₂ animals (FIG. 6B), although these datadid not reach statistical significance. CI-1040, however, significantlyreduced the levels of circulating ELT3 cells in the blood of E₂-treatedanimals by 84% (P=0.042, FIG. 6C). Most strikingly, no lung metastaseswere detected in mice treated with E₂ plus CI-1040 (P=0.046, FIGS. 6Dand E).

To investigate further the role of MEK/ERK on the survival of ELT3 cellsin the circulation, ELT3-luciferase cells were intravenously injectedinto mice treated with E₂ along or E₂ plus CI-1040. At 2 h post-cellinjection, similar levels of bioluminescence were observed in the chestregions of all mice. At 5 h, the bioluminescence in the chest regions ofthe E₂ plus CI-1040 treated mice was decreased by 55%, as compared tothat in the E₂-treated mice (P=0.02, FIG. 6F). After sacrifice at 60 hpostcell injection, the bioluminescent signals in the ex vivo lungs ofthe E₂ plus CI-1040-treated mice were significantly reduced by 96%, ascompared to the signals in the E₂-treated animals (P=0.0045, FIG. 6F).

It will be appreciated from the foregoing results that estrogentreatment of both female and male mice bearing Tsc2-null ELT3 xenografttumors results in an increase in pulmonary metastases. Theestrogen-driven metastasis of ELT3 cells was associated with activationof p42/44 MAPK both in vitro and in vivo. Treatment of the mice with theMEK1/2 inhibitor CI-1040 completely blocked the lung metastases inestrogen-treated animals, while causing only a 25% reduction in the sizeof the primary xenograft tumors, indicating that activation of MEK by E₂is a critical factor in the metastasis of Tsc2-null cells. In contrastto CI-1040, the mTOR inhibitor RAD001 completely blocked formation ofthe primary tumor.

Estrogen is known to activate the MAPK pathway (Magliaccio, A. et al.,EMBO J., 15: 1292-1300 (1996); Razandi, M. et al., J. Biol. Chem., 278:2701-2712 (2003); Song, R. X. et al., Mol. Endocrinol., 16: 116-127(2002); Song, R. X. et al., Endocrinology, 148: 4091-4101 (2007)). It ishypothesized that tuberin-null cells may be particularly sensitive toactivation of the Raf/MEK/MAPK signaling cascade by estrogen, because atbaseline this signaling pathway is inhibited by Rheb, the target oftuberin's GTPase activating protein domain (Im, E. et al., supra;Karbowniczek, M. et al., supra (2004); Karbowniczek, M., supra (2006)).Metastasis is a complex process, and there are numerous mechanismsthrough which estrogen's activation of MEK may enhance the metastasis ofTsc2-null cells. The in vitro studies described herein revealed thatestrogen induces resistance to anoikis in Tsc2-null cells, whichsuggests that one of these mechanisms involves the survival of detachedcells. This is consistent with our finding of markedly elevated levelsof circulating tumor cells in estrogen-treated mice bearing xenografttumors. We also found that estrogen treatment enhances the survival ofintravenously injected cells in the peripheral blood. These data are ofparticular interest because circulating LAM cells can be detected in theblood and pleural fluid of women with LAM (Crooks, D. M. et al., supra).Our data provide a rationale for the potential use of circulating cellsas a quantitative and rapid biomarker of response to targeted therapy inwomen with LAM.

In addition to promoting the levels of ELT3 cells in the peripheralblood, as measured by real-time RT-PCR using rat-specific primers,estrogen also enhanced the survival of intravenously injectedluciferase-expressing ELT3 cells within the lungs. Three hours afterinjection, there was significantly more bioluminescence in the chestregions of the E₂-treated animals, and by 24 h this differences was evenmore marked. Importantly, however, 1 h after the i.v. injection ofELT3-luciferase cells, similar levels of bioluminescence were present inthe lungs of estrogen-treated and placebo-treated animals, whichdemonstrates that similar numbers of injected cells reach the lungs.These data suggest that E₂ promotes the survival of Tsc2-null cellswithin the lungs.

The lack of an in vivo model of LAM has been a significant barrier inLAM research. While not a perfect surrogate, ELT3 cells have importantfeatures in common with LAM cells, including loss of Tsc2, activation ofmTOR, and expression of estrogen receptor alpha and smooth musclemarkers (Howe, S. R. et al., Am. J. Pathol., supra (1995); Howe, S. R.,Endocrinology, supra (1995)).

The animal model described herein provides a useful tool for screeningcandidate inhibitors that target signaling pathways andhormonally-driven events.

A number of patent and non-patent publications are cited throughout theforegoing specification in order to describe the state of the art towhich this invention pertains. The entire disclosure of each of thesepublications is incorporated by reference herein.

While certain embodiments of the present invention have been describedand/or exemplified above, various other embodiments will be apparent tothose skilled in the art from the foregoing specification. For example,the compounds of Formula I, above, may be effective for the treatment ofother estrogen-mediated malignancies, such as breast or ovarian cancer.The present invention is, therefore, not limited to the particularembodiments described and/or exemplified, but is capable of considerablevariation and modification without departure from the scope of theappended claims.

What is claimed is:
 1. A method for identifying agents having efficacyfor the treatment or prevention of LAM, comprising: a) generating axenograft mouse model of LAM via introduction of a sufficient number ofELT3 cells into a scid mouse thereby forming tumors having metastaticcapability; and b) administering a test compound to said mice anddetermining whether said compound alters tumor size or inhibitsmetastatic capability thereof.
 2. The method of claim 1 furthercomprising the step of determining whether said agent induces anoikis insaid ELT3 cells.
 3. A method for the treatment or prophylaxis of LAMcomprising administering to a patient in need thereof a therapeuticallyeffective amount of RAD001.
 4. A xenograft animal model of LAM obtainedby introducing into a scid mouse a sufficient number of ELT3 cells toform tumors having metastatic capability.
 5. The animal model of claim4, wherein said ELT3 cells are subcutaneously injected into the flanksof said mouse.
 6. The animal model of claim 4, wherein said ELT3 cellsare injected into a tail vein of said mouse.